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Abstract Ionizing radiation has been pivotal in cancer therapy since its discovery. Despite its therapeutic benefits, IR causes significant acute and chronic complications due to DNA damage and the generation of reactive oxygen species, which harm nucleic acids, lipids, and proteins. While cancer cells are more vulnerable to ionizing radiation due to their inefficiency in repairing damage, healthy cells in the irradiated area also suffer. Various types of cell death occur, including apoptosis, necrosis, pyroptosis, autophagy-dependent cell death, immunogenic cell death, and ferroptosis. Ferroptosis, driven by iron-dependent lipid peroxide accumulation, has been recognized as crucial in radiation therapy’s therapeutic effects and complications, with extensive research across various tissues. This review aims to summarize the pathways involved in radiation-related ferroptosis, findings in different organs, and drugs targeting ferroptosis to mitigate its harmful effects.

Abstract Background Radiation-induced fibrosis (RIF) is an important late complication of radiation therapy, and the resulting damaging effects of RIF can significantly impact reconstructive outcomes. There is currently a paucity of effective treatment options available, likely due to the continuing knowledge gap surrounding the cellular mechanisms involved. In this study, detailed analyses of irradiated and non-irradiated human skin samples were performed incorporating histological and single-cell transcriptional analysis to identify novel features guiding development of skin fibrosis following radiation injury. Methods Paired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing. Results Comparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0–4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses. Conclusion In response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.

Abstract Local skin flaps are frequently employed for wound closure to address surgical, traumatic, congenital, or oncologic defects. (1) Despite their clinical utility, skin flaps may fail due to inadequate perfusion, ischemia/reperfusion injury (IRI), excessive cell death, and associated inflammatory response. (2) All of these factors contribute to skin flap necrosis in 10–15% of cases and represent a significant surgical challenge. (3, 4) Once flap necrosis occurs, it may require additional surgeries to remove the entire flap or repair the damage and secondary treatments for infection and disfiguration, which can be costly and painful. (5) In addition to employing appropriate surgical techniques and identifying healthy, well-vascularized tissue to mitigate the occurrence of these complications, there is growing interest in exploring cell-based and pharmacologic augmentation options. (6) These agents typically focus on preventing thrombosis and increasing vasodilation and angiogenesis while reducing inflammation and oxidative stress. Agents that modulate cell death pathways such as apoptosis and autophagy have also been investigated. (7) Implementation of drugs and cell lines with potentially beneficial properties have been proposed through various delivery techniques including systemic treatment, direct wound bed or flap injection, and topical application. This review summarizes pharmacologic- and cell-based interventions to augment skin flap viability in animal models, and discusses both translatability challenges facing these therapies and future directions in the field of skin flap augmentation.

Foreign body response (FBR) is a universal reaction to implanted biomaterial that can affect the function and longevity of the implant. A few studies have attempted to identify targets for treating FBR through the use of single-cell RNA sequencing (scRNA-seq), though the generalizability of these findings from an individual study may be limited. In our study, we perform a meta-analysis of scRNA-seq data from all available FBR mouse studies and integrate these data to identify gene signatures specific to FBR across different models and anatomic locations. We identify subclusters of fibroblasts and macrophages that emerge in response to foreign bodies and characterize their signaling pathways, gene ontology terms, and downstream mediators. The fibroblast subpopulations enriched in the setting of FBR demonstrated significant signaling interactions in the transforming growth factor-beta (TGF-β) signaling pathway, with known pro-fibrotic mediators identified as top expressed genes in these FBR-derived fibroblasts. In contrast, FBR-enriched macrophage subclusters highly expressed pro-fibrotic and pro-inflammatory mediators downstream of tumor necrosis factor (TNF) signaling. Cell–cell interactions were additionally interrogated using CellChat, with identification of key signaling interactions enriched between fibroblasts and macrophages in FBR. By combining multiple FBR datasets, our meta-analysis study identifies common cell-specific gene signatures enriched in foreign body reactions, providing potential therapeutic targets for patients requiring medical implants across a myriad of devices and indications.

Abstract Mandibular distraction osteogenesis (DO) is mediated by skeletal stem cells (SSCs) in mice, which enact bone regeneration via neural crest re-activation. As peripheral nerves are essential to progenitor function during development and in response to injury, we questioned if denervation impairs mandibular DO. C57Bl6 mice were divided into two groups: DO with a segmental defect in the inferior alveolar nerve (IAN) at the time of mandibular osteotomy (“DO Den”) and DO with IAN intact (“DO Inn”). DO Den demonstrated significantly reduced histological and radiological osteogenesis relative to DO Inn. Denervation preceding DO results in reduced SSC amplification and osteogenic potential in mice. Single cell RNA sequencing analysis revealed that there was a predominance of innervated SSCs in clusters dominated by pathways related to bone formation. A rare human patient specimen was also analyzed and suggested that histological, radiological, and transcriptional alterations seen in mouse DO may be conserved in the setting of denervated human mandible distraction. Fibromodulin (FMOD) transcriptional and protein expression were reduced in denervated relative to innervated mouse and human mandible regenerate. Finally, when exogenous FMOD was added to DO-Den and DO-Inn SSCs undergoing in vitro osteogenic differentiation, the osteogenic potential of DO-Den SSCs was increased in comparison to control untreated DO-Den SSCs, modeling the superior osteogenic potential of DO-Inn SSCs.

Background:. As visibility of the transgender patient population and utilization of online resources increases, it is imperative that web-based gender-affirming surgery (GAS) materials for patients are readable, accessible, and of high quality. Methods:. A search trends analysis was performed to determine frequency of GAS-related searches over time. The top 100 most common results for GAS-related terms were analyzed using six readability formulas. Accessibility of patient-facing GAS sources was determined by categorizing types of search results. Frequency of article types was compared in low- and high-population dense areas. Quality was assigned to GAS web-based sources using the DISCERN score. Results:. Search engine trend data demonstrates increasing occurrence of searches related to GAS. Readability scores of the top 100 online sources for GAS were discovered to exceed recommended levels for patient proficiency. Availability of patient-facing online information related to GAS was found to be 60%, followed by information provided by insurance companies (17%). Differences in availability of online resources in varying dense cities were found to be minimal. The average quality of sources determined by the DISCERN score was found to be 3, indicating “potential important shortcomings.” Conclusions:. Despite increasing demand for web-based GAS information, the readability of online resources related to GAS was found to be significantly greater than the grade level of proficiency recommended for patients. A high number of nonpatient-facing search results appear in response to GAS search terms. Quality sources are still difficult for patients to find, as search results have a high incidence of low-quality resources.

Abstract Background Radiation-induced skin injury is a well-known risk factor for impaired wound healing. Over time, the deleterious effects of radiation on skin produce a fibrotic, hypovascular dermis poorly suited to wound healing. Despite increasing understanding of the underlying pathophysiology, therapeutic options remain elusive. Deferoxamine (DFO), an iron-chelating drug, has been shown in prior murine studies to ameliorate radiation-induced skin injury as well as improve wound healing outcomes in various pathologic conditions when administered transdermally. In this preclinical study, we evaluated the effects of deferoxamine on wound healing outcomes in chronically irradiated murine skin. Methods Wild-type mice received 30 Gy of irradiation to their dorsal skin and were left to develop chronic fibrosis. Stented excisional wounds were created on their dorsal skin. Wound healing outcomes were compared across 4 experimental conditions: DFO patch treatment, vehicle-only patch treatment, untreated irradiated wound, and untreated nonirradiated wounds. Gross closure rate, wound perfusion, scar elasticity, histology, and nitric oxide assays were compared across the conditions. Results Relative to vehicle and untreated irradiated wounds, DFO accelerated wound closure and reduced the frequency of healing failure in irradiated wounds. DFO augmented wound perfusion throughout healing and upregulated angiogenesis to levels observed in nonirradiated wounds. Histology revealed DFO increased wound thickness, collagen density, and improved collagen fiber organization to more closely resemble nonirradiated wounds, likely contributing to the observed improved scar elasticity. Lastly, DFO upregulated inducible nitric oxide synthase and increased nitric oxide production in early healing wounds. Conclusion Deferoxamine treatment presents a potential therapeutic avenue through which to target impaired wound healing in patients following radiotherapy.

Implantable biomaterials represent the forefront of regenerative medicine, providing platforms and vessels for delivering a creative range of therapeutic benefits in diverse disease contexts. However, the chronic damage resulting from implant rejection tends to outweigh the intended healing benefits, presenting a considerable challenge when implementing treatment-based biomaterials. In response to implant rejection, proinflammatory macrophages and activated fibroblasts contribute to a synergistically destructive process of uncontrolled inflammation and excessive fibrosis. Understanding the complex biomaterial–host cell interactions that occur within the tissue microenvironment is crucial for the development of therapeutic biomaterials that promote tissue integration and minimize the foreign body response. Recent modifications of specific material properties enhance the immunomodulatory capabilities of the biomaterial and actively aid in taming the immune response by tuning interactions with the surrounding microenvironment either directly or indirectly. By incorporating modifications that amplify anti-inflammatory and pro-regenerative mechanisms, biomaterials can be optimized to maximize their healing benefits in harmony with the host immune system.

Wound healing results in the formation of scar tissue which can be associated with functional impairment, psychological stress, and significant socioeconomic cost which exceeds 20 billion dollars annually in the United States alone. Pathologic scarring is often associated with exaggerated action of fibroblasts and subsequent excessive accumulation of extracellular matrix proteins which results in fibrotic thickening of the dermis. In skin wounds, fibroblasts transition to myofibroblasts which contract the wound and contribute to remodeling of the extracellular matrix. Mechanical stress on wounds has long been clinically observed to result in increased pathologic scar formation, and studies over the past decade have begun to uncover the cellular mechanisms that underly this phenomenon. In this article, we will review the investigations which have identified proteins involved in mechano-sensing, such as focal adhesion kinase, as well as other important pathway components that relay the transcriptional effects of mechanical forces, such as RhoA/ROCK, the hippo pathway, YAP/TAZ, and Piezo1. Additionally, we will discuss findings in animal models which show the inhibition of these pathways to promote wound healing, reduce contracture, mitigate scar formation, and restore normal extracellular matrix architecture. Recent advances in single cell RNA sequencing and spatial transcriptomics and the resulting ability to further characterize mechanoresponsive fibroblast subpopulations and the genes that define them will be summarized. Given the importance of mechanical signaling in scar formation, several clinical treatments focused on reducing tension on the wound have been developed and are described here. Finally, we will look toward future research which may reveal novel cellular pathways and deepen our understanding of the pathogenesis of pathologic scarring. The past decade of scientific inquiry has drawn many lines connecting these cellular mechanisms that may lead to a map for the development of transitional treatments for patients on the path to scarless healing.

Summary: Despite its rapidly increased availability for the study of complex tissue, single-cell RNA sequencing remains prohibitively expensive for large studies. Here, we present a protocol using oligonucleotide barcoding for the tagging and pooling of multiple samples from healing wounds, which are among the most challenging tissue types for this application. We describe steps to generate skin wounds in mice, followed by tissue harvest and oligonucleotide barcoding. This protocol is also applicable to other species including rats, pigs, and humans.For complete details on the use and execution of this protocol, please refer to Stoeckius et al. (2018),1 Galiano et al. (2004),2 and Mascharak et al. (2022).3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Background:. Distraction osteogenesis (DO) is used for skeletal defects; however, up to 50% of cases exhibit complications. Previous mouse models of long bone DO have been anecdotally hampered by postoperative complications, expense, and availability. To improve clinical techniques, cost-effective, reliable animal models are needed. Our focus was to develop a new mouse tibial distractor, hypothesized to result in successful, complication-free DO. Methods:. A lightweight tibial distractor was developed using CAD and 3D printing. The device was fixed to the tibia of C57Bl/6J mice prior to osteotomy. Postoperatively, mice underwent 5 days latency, 10 days distraction (0.15 mm every 12 hours), and 28 days consolidation. Bone regeneration was examined on postoperative day 43 using micro-computed tomography (μCT) and Movat’s modified pentachrome staining on histology (mineralized volume fraction and pixels, respectively). Costs were recorded. We compared cohorts of 11 mice undergoing sham, DO, or acute lengthening (distractor acutely lengthened 3.0 mm). Results:. The histological bone regenerate was significantly increased in DO (1,879,257 ± 155,415 pixels) compared to acute lengthening (32847 ± 1589 pixels) (P < 0.0001). The mineralized volume fraction (bone/total tissue volume) of the regenerate was significantly increased in DO (0.9 ± 0.1) compared to acute lengthening (0.7 ± 0.1) (P < 0.001). There was no significant difference in bone regenerate between DO and sham. The distractor was relatively low cost ($11), with no complications. Conclusions:. Histology and µCT analysis confirmed that the proposed tibial DO model resulted in successful bone formation. Our model is cost-effective and reproducible, enabling implementation in genetically dissectible transgenic mice.

Wound healing is the body’s process of injury recovery. Skin healing is divided into four distinct overlapping phases: hemostasis, inflammation, proliferation, and remodeling. Cell-to-cell interactions mediated by both cytokines and chemokines are imperative for the transition between these phases. Patients can face difficulties in the healing process due to the wound being too large, decreased vascularization, infection, or additional burdens of a systemic illness. The field of tissue engineering has been investigating biomaterials as an alternative for skin regeneration. Biomaterials used for wound healing may be natural, synthetic, or a combination of both. Once a specific biomaterial is selected, it acts as a scaffold for skin regeneration. When the scaffold is applied to a wound, it allows for the upregulation of distinct molecular signaling pathways important for skin repair. Although tissue engineering has made great progress, more research is needed in order to support the use of biomaterials for wound healing for clinical translation.

Humans and other large mammals heal wounds by forming fibrotic scar tissue with diminished function. Here, the authors show that disrupting mechanotransduction through the focal adhesion kinase pathway in large animals accelerates healing, prevents fibrosis, and enhances skin regeneration.

Pancreatic cancer is one of the deadliest forms of cancer with one of the lowest 5-year survival rates of all cancer types. A defining characteristic of pancreatic cancer is the existence of dense desmoplastic stroma that, when exposed to stimuli such as cytokines, growth factors, and chemokines, generate a tumor-promoting environment. Cancer-associated fibroblasts (CAFs) are activated during the progression of pancreatic cancer and are a crucial component of the tumor microenvironment (TME). CAFs are primarily pro-tumorigenic in their activated state and function as promoters of cancer invasion, proliferation, metastasis, and immune modulation. Aided by many signaling pathways, cytokines, and chemokines in the tumor microenvironment, CAFs can originate from many cell types including resident fibroblasts, mesenchymal stem cells, pancreatic stellate cells, adipocytes, epithelial cells, endothelial cells, and other cell types. CAFs are a highly heterogeneous cell type expressing a variety of surface markers and performing a wide range of tumor promoting and inhibiting functions. Single-cell transcriptomic analyses have revealed a high degree of specialization among CAFs. Some examples of CAF subpopulations include myofibrotic CAFs (myCAFs), which exhibit a matrix-producing contractile phenotype; inflammatory CAFs (iCAF) that are classified by their immunomodulating, secretory phenotype; and antigen-presenting CAFs (apCAFs), which have antigen-presenting capabilities and express Major Histocompatibility Complex II (MHC II). Over the last several years, various attempts have been undertaken to describe the mechanisms of CAF–tumor cell interaction, as well as CAF–immune cell interaction, that contribute to tumor proliferation, invasion, and metastasis. Although our understanding of CAF biology in cancer has steadily increased, the extent of CAFs heterogeneity and their role in the pathobiology of pancreatic cancer remains elusive. In this regard, it becomes increasingly evident that further research on CAFs in pancreatic cancer is necessary.

As a basic science, craniofacial research embraces multiple facets spanning from molecular regulation of craniofacial development, cell biology/signaling and ultimately translational craniofacial biology. Calvarial sutures coordinate development of the skull, and the premature fusion of one or more, leads to craniosynostosis. Animal models provide significant contributions toward craniofacial biology and clinical/surgical treatments of patients with craniofacial disorders. Studies employing mouse models are costly and time consuming for housing/breeding. Herein, we present the establishment of a calvarial suture explant 2-D culture method that has been proven to be a reliable system showing fidelity with the in vivo harvesting procedure to isolate high yields of skeletal stem/progenitor cells from small number of mice. Moreover, this method allows the opportunity to phenocopying models of craniosynostosis and in vitro tamoxifen-induction of ActincreERT2;R26Rainbow suture explants to trace clonal expansion. This versatile method tackles needs of large number of mice to perform calvarial suture research.

Abstract Background Children born with a small or absent ear undergo surgical reconstruction to create a suitable replacement using rib cartilage. To overcome the donor site morbidity and long-term pain of harvesting rib cartilage, synthetic materials can be a useful alternative. Medpor, is the currently used synthetic polyethylene material to replace missing facial cartilage but unfortunately it has high levels of surgical complications including infection and extrusion, making it an unsuitable replacement. New materials for facial cartilage reconstruction are required to improve the outcomes of surgical reconstruction. This study has developed a new nanomaterial with argon surface modification for auricular cartilage replacement to overcome the complications with Medpor. Results Polyurethanes nanocomposites scaffolds (PU) were modified with argon plasma surface modification (Ar) and compared to Medpor in vitro and in vivo. Ar scaffolds allowed for greater protein adsorption than Medpor and PU after 48 h (p

Purpose: Scarring in the mouse dorsal dermis is mediated by pro-fibrotic, Engrailed-1 lineage-positive fibroblasts (EPFs). We recently showed that mechanotransduction blockade (YAP inhibition, using the drug verteporfin), results in complete wound regeneration, with full recovery of normal dermal appendages (hair follicles, glands), extracellular matrix (ECM) architecture, and tensile strength. This regenerative outcome following verteporfin treatment is mediated by Engrailed-1 lineage-negative fibroblasts (ENFs). The complex milieu of cell types and molecular signals involved in wound repair makes it difficult to study using any single data modality. Thus, we sought to use a holistic approach, incorporating multiple high-throughput, high-dimensional analyses, to define the divergent molecular events distinguishing typical scarring healing from verteporfin-induced wound regeneration. Methods: C57BL/6J mice underwent dorsal splinted excisional wounding per standard protocol. Wounds were treated with local injection of either verteporfin or vehicle control (PBS) on POD 0. We harvested unwounded skin and wounds at POD 2, 7, 14, and 30 (n=5 mice/timepoint and treatment) and subjected wound cells to three analyses: single-cell RNA-sequencing (scRNA-seq, using 10X Genomics Chromium); timsTOF, a recently-developed, high-throughput proteomic sequencing platform; and a novel machine learning algorithm for quantitatively comparing ECM ultrastructure. Results: Pseudotime analysis (Monocle3) of pooled scRNA-seq data revealed that fibroblasts followed two distinct transcriptional trajectories, one characterized by mechanical activation (En-1 lineage-positive, “fibrotic” trajectory) and the other characterized by developmental and regenerative pathways (En-1 lineage-negative; Rspo1, Dkk2/3, Trps1). Cross-platform data integration confirmed that fibroblasts in the fibrotic trajectory correlated with myofibroblast proteomic signatures (Col1a1/2, Fn1, etc.) and fibrotic/scar ECM features. In contrast, fibroblasts in the regenerative trajectory negatively correlated with myofibroblast markers and were associated with a “basket-weave” ECM pattern quantitatively indistinguishable from that of unwounded skin. Our integrated dataset suggested an important role for Wnt pathway proteins in ENF-mediated skin regeneration, so we compared POD 14 scars and regenerated wounds by multiplexed in situ hybridization (RNAScope) for Rspo1 (Wnt agonist), Trps1 (master hair follicle regulator), Ank1 (YAP target gene), and Dpp4 (EPF marker). Quantification of RNA granules across thousands of cells using a custom image analysis pipeline revealed that ENF-mediated healing (low Dpp4) in YAP-inhibited (low Ank1) wounds yielded regeneration of functional hair follicles through Wnt-mediated pathway activation (high Rpos1, Trps1). These data suggest that YAP inhibition unlocks wound regeneration via Wnt-active, En-1 lineage-negative fibroblasts. Conclusion: By studying regenerating (verteporfin-treated) versus scarring wounds across multiple healing timepoints and high-dimensional data modalities, we were able to profile fibrotic versus regenerative healing at unprecedented depth. Our integrated analysis revealed that dermal fibroblasts in these two wound settings exhibit distinct molecular trajectories defined by divergent transcriptomic, proteomic, and ultrastructural properties. Further, we found that wound regeneration in the context of verteporfin treatment is associated with suppression of mechanical signaling and activation of key Wnt pathway members including Trps1 (a gene with known hair follicle developmental roles). These results could have important implications for both the fundamental study of wound healing and potential anti-scarring therapeutic avenues